It is now known that rheumatoid arthritis (RA)-related autoantibodies, including autoantibodies to citrullinated peptide/protein antigens (ACPA) and rheumatoid factors (RF), are found in asymptomatic individuals for an average of 3-5 years prior to the development of clinically apparent arthritis. This autoantibody positive but asymptomatic phase is characterized by progressive epitope spreading and the development of increased systemic inflammation, ultimately culminating in classified RA. The understanding of this process has progressed such that an NIH-funded, double blind, placebo controlled prevention trial in very high risk asymptomatic ACPA+ individuals has been initiated. The investigative group for this application now proposes to focus efforts on understanding the molecular mechanisms that underlie the initiation, development and evolution of RA-related autoimmunity in this preclinical period, with the goals of identifying the inciting antigen(s) and developing tolerance strategies that could be utilized in these early disease phases. As outlined within the proposal, ongoing studies have strongly suggested that the immune process ultimately manifesting clinically as RA is initiated at mucosal sites and is associated with local inflammation and local autoantibody production, which in a subset of individuals evolves to become systemic autoimmunity detected through blood- based autoantibody biomarkers. Major preliminary findings have included that the peripheral blood plasmablast pool in the at-risk population is uniquely characterized by expanded IgA isotype expressing cells and dual IgA/IgG clonal families. Additionally, sampling the lung and gut mucosa in at-risk subjects through studies of sputum and feces, respectively, has revealed that an elevated proportion of individuals locally express IgA ACPA, and only a small subset of bacterial families is specifically recognized by locally produced IgA. With these findings, it should now be possible to identify the citrullinated epitopes to which pathogenic B and T cells respond locally and systemically, as well as understand how local mucosal immune responses to a subset of bacteria influence development of RA-related autoimmunity. This knowledge is critical to our understanding of the immunologic mechanisms that underlie disease development as well as the design of tolerance-inducing therapies. Notably, the Co-Investigators in this CSGADP application, Drs. Kuhn, Robinson and Buckner, have developed novel approaches to characterize the mucosal immune response and also identify antigen-specific B and T cell responses to citrullinated and other RA-related autoantigens. To advance our understanding of the immune and autoimmune responses at these earliest time points, the investigators propose to utilize these techniques in at-risk subjects to characterize the antigen reactivity of dual IgA/IgG plasmablasts and mucosally produced IgA/IgG, determine which citrullinated antigens and microbial species are recognized, understand how clonotypes and effector function of individual citrullinated antigen-specific clonal families in B cells evolve to be increasingly pathogenic, and characterize CD4+ T cells that recognize citrullinated and novel antigens.